1. Field of The Invention
The present invention relates to electrophoresis, particularly capillary electrophoresis, and more particularly capillary gel electrophoresis.
2. Description of Related Art
Recently, there has been a great deal of activity in DNA analysis by capillary electrophoretic methods ([1] Cohen, A. S.; Najarian, D. R.; Paulus, A; Guttman, A; Smith, J. A.; Karger, B. L.; Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 9660-9663; [2] Paulus, A., Gassmann, E.; Field, M. J.; Electrophoresis, 1990, 11, 702-708; [3] Yin, H. F.; Lux, J. A.; Shomburg, G.; J. High Res. Chrom., 1990, 13, 624-627.) Using this new and powerful technology, results have been published on the separation of single-stranded DNA molecules, such as in synthetic DNA analysis ([4] Guttman, A.; Cohen, A. S.; Heiger, D. N.; Karger, B. L.; Anal. Chem., 1990, 62, 2038-2042), as well as in the separation of double-stranded DNA molecules, particularly concerning PCR products and restriction fragments ([5] Schwartz, H. E.; Ulfelder, K.; Sunzeri, F.; Busch, M.; Brownlee, M.G.; J. Chromatogr. 1991, 559, 267-283.) It was also demonstrated that high-efficiency, fast separations of DNA molecules can be achieved by the use of linear polyacrylamide gel-filled capillary columns ([6] Heiger, D. N.; Cohen, A. S.; Karger, B. L.; J. Chromatogr., 1990, 516, 33-48; [7] Guttman, A.; Cooke, N.; J. Chromatogr., 1991, 559, 285-294.) Even fragments of the same chain length with different sequences were separated by this method due to differences in molecular conformation ([8] Guttman, A.; Nelson, R. J.; Cooke, N. J. Chromatogr., 1991, 593, 297-303.)
Different field operation techniques have been described recently to achieve better separation of different size DNA molecules, mainly with slab gel electrophoresis. Dennison et al ([9] Dennison, C.; Linder, W. A.; Phillis, N. C. K.; Anal. Biochem, 1982, 120, 12-18) employed conical or wedge-shaped slab gels to linearize the logarithmic distribution of bands (nonlinear voltage gradient method) Biggin et al ([10] Biggin, M. D.; Gibson, T. J.; Hong, G. F.; Proc. Natl. Acad. Sci. U.S.A., 1983, 80, 3963-3965) studied the usefulness of high ionic strength anode buffer where the resistance of the gel in the direction of the anode decreases and creates a negative field- strength gradient along the DNA's migration path. They concluded that this particular method is not practical for ultrathin gels, as is also the case for capillary gel electrophoresis ([11] Guttman, A.; Beckman Instruments, Inc., Research and Development, Unpublished results, 1991). Ansorge et al tried using an increasing cross-sectional area of the slab gels producing a field gradient to achieve enhanced sharpening of bands, thereby increasing the number of resolvable bases per gel ([12] Ansorge, W.; Labeit, S.; J. Biochem. Biophys. Methods, 1984, 10, 237-243.) Cantor et al ([13] Cantor, C. R.; Smith, C. L.; Mathew, M. K.; Ann. Rev. Biophys. Biophys. Chem., 1988, 17, 287-304) introduced the pulsed field method (changing the direction and magnitude of the field in an oscillating manner), that takes advantage of the elongated and oriented configuration of large DNA (&lt;50 kbp) molecules in gels. Heiger et al [6] described the capillary gel electrophoretic separation of double-stranded DNA molecules up to 23,000 base pairs in size using the pulsed field technique with very low gel concentrations. Although in slab gel operation there are some mechanical difficulties involved in handling low concentration gels ([14] Fangman, W. L.; Nucl. Acid Res., 1978, 5, 653-665), this does not present a problem in capillary electrophoresis techniques. Demana and co-workers ([15] Demana, T.; Lanan, M.; Morris, M. D.; Anal. Chem., 1991, 63, 2795-2797) used an analyte velocity modulation method to increase separation power in capillary gel electrophoresis of DNA restriction fragments.